Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Genital tract infections with human papillomaviruses (HPVs) are manifested in many ways, from an asymptomatic carrier state, to the commonly recognized disease condylomata acuminata (genital warts), and to overt malignancy. Infections with certain genital HPVs, such as HPV types 6 and 11 cause external genital warts, a condition characterized by marked epithelial proliferation and a low risk of dysplastic changes. In contrast, other genital HPVs including types 16, 18, and 31 are highly associated with dysplastic cervical lesions. These dysplasia-associated, or "high risk" HPV types can be detected in external genital warts removed from immunosuppressed patients. About half of the lesions from immunosuppressed patients contain a high risk HPV type, often in a mixed infection with a "low risk" type such as HPV type 6 or 11.
For an HPV to be assigned a type number, the entire genome must be cloned and three gene regions (E6, E7, and L1) must be sequenced and compared to known types. With the advent of consensus primer PCR amplification for detection of HPV DNA in clinical specimens, it has become clear that there are a significant number of HPVs in addition to the numbered types. The most commonly used consensus PCR system uses degenerate primers located in a conserved region of the L1 (major capsid) gene to amplify a segment of about 450 base pairs. A number of HPV sequences have been found which do not match well with any of the known (numbered) HPV types. These are usually designated by an arbitrary name assigned by the group that first recognized it. In some cases, the same or closely related sequences are independently reported by different groups and given different names. While these almost certainly represent novel HPV types, they are designated by these "unconventional" names until the full genome is cloned and characterized. Several of the uncharacterized HPVs have been identified in specimens from immunocompromised patients.
One such partially characterized HPV is HPV MM7 (also called Pap 291) (Manos, M. M., Waldman, J., Zhang, T. Y., Greer, C. E., Eichinger, G., Schiffman, M. H., and Wheeler, C. M. (1994) J. Infectious Diseases 170(5), 1096-1099). This virus type is closely related to HPV LVX82 (Ong, C. K., Bernard, H. U., and Villa, L. L. (1994) J. Infectious Diseases 170(5), 1086-1088), and the two are sometimes referred to as HPV MM7/LVX82. Infection with this HPV type appears to be relatively common compared to other genital HPVs. All published studies that have identified HPV MM7/LVX82 have been performed on cervical lavage or cervical biopsy samples rather than in DNA extracted from genital warts. In one study, HPV MM7/LVX82 was identified in 20 of 338, or 5.9% of cervical samples from patients with low grade dysplasia, and 1 of 43, or 2.3% of high grade dysplasia samples. In a study of cervical HPV acquisition by college women, incident infection with HPV MM7/LVX82 (called Pap 291 in this study) was identified in 15 of 529, or 2.8% of women followed over a period of two years, making it one of the more common types acquired (Ho, G. Y. F., Bierman, R., Beardsley, L., Chang, C. J., and Burk, R. D. (1998) New England J. Med. 338(7), 423-428).
In another study, 22 HPV types were identified in cervical lavage samples of human immunodeficiency virus (HIV)-seropositive women, and seven types in lavage samples from HIV-seronegative women (Shah K. V., Solomon, L., Daniel, R., Cohn, S., and Vlahov, D. (1997) J. Clin. Microbiol. 35(2), 517-519). Overall, 104 of 150, or 69.3% of HIV-seropositive women had detectable HPV compared to 19 of 72, or 26.4% of HIV-seronegative women. The most prevalent HPV type detected in both groups of women was HPV MM7/LVX82, accounting for 20 of 150, or 13.3% of HIV-seropositive women and five of 72, or 6.9% of HIV-seronegative women. Of the 123 women with detectable HPV of any type, HPV MM7/LVX82 was the most common type detected, present in 20% of samples.
The development of vaccines against human papillomaviruses (HPV) has been hampered by the lack of a conventional culture system because HPV completes its life cycle only in fully differentiated human tissue. To overcome this obstacle, the athymic mouse xenograft system has been used to study the pathogenesis of HPV 11, and to attempt to develop neutralizing assays for vaccine development (Kreider, J. W., M. K. Howett, A. E. Leure-Dupree, R. J. Zaino and J. A. Weber. (1987) J. Virol. 61, 590-593; Kreider, J. W., Howlett, M. K., Lill, N. L., Bartlett, G. L., Zaino, R. J., Sedlacek, T. V., and Mortel, R. (1986) J. Virol. 59, 369-376). In this method, an extract from HPV-infected tissue is used to infect human foreskin fragments, which are then implanted under the renal capsules of athymic mice. After ascertaining that an implant is HPV infected, the virus contained in the implant is propagated by additional rounds of xenograft implantation into athymic mice. Recently, HPV 40 has been produced in this system (Christensen N. D., Koltun, W. A., Cladel, N. M., Budgeon, L. R., Reed, C. A., Kreider, J. W., Welsh, P. A., Patrick, S. D., and Yang, H. (1997) J. Virol. 71(10), 7337-7344). Production of an isolate consistent with HPV MM7/LVX82 was also reported. However, an infectious stock of HPV MM7/LVX82 was not obtained. HPV 16 has been produced using a similar system, implanting into mice with severe combined immune deficiency (SCID).
From the foregoing discussion, it is clear that live, infectious stocks of a variety of types of human papillomavirus are needed in order to produce and test broad spectrum vaccines against these viruses. It is particularly important that such a vaccine is targeted to commonly found HPV types, such as the MM7/LVX82. At present, only a small number of HPV types, HPV 11, HPV 40 and HPV 16, are available as infectious stocks. Accordingly, it is an object of the invention to provide a live, infectious stock of HPV MM7/LVX82. It is a further object of the present invention to provide vaccines directed against HPV MM7, HPV LVX82 and similar types, as well as methods for testing the ability of those vaccines to elicit an appropriate immune response against the selected HPV types.